Two significant problems are encountered in recombinant peptide expression. First, many biologically active peptides have an amide at their C-terminus. Such amidated peptides are not typically produced through recombinant expression. Second, C-terminal amide group substitution, performed routinely in vivo, proves difficult to perform in vitro.
It is well known that the production of short and medium range peptides of less than about 100 amino acids in length by expression of peptide-encoding DNA in a recombinant host cell such as E. coli is commonly plagued by the problem of enzymatic degradation of the expressed peptide within the host cell, resulting in partial or complete loss of the peptide. The most commonly employed means to overcome this problem is to insolubilize the peptide within the host cell. This can be affected by expressing the peptide as a chimeric Normally, the fusion partner will be fused to the N-terminus of the peptide. The chimeric protein forms inclusion bodies within the cell, within which the peptide is protected from degradation by proteolytic enzymes.
Once the inclusion bodies are recovered from the host cell, the peptide must be separated from the leader sequence, purified and recovered in an active form. Separation from the leader sequence may be accomplished by placing a sequence of amino acids at the junction of the leader and the peptide which are specifically recognized and cleaved under appropriate conditions, e.g. acid cleavage or enzymatic cleavage.
For example, introduction of acid-labile aspartyl-proline linkage between the two segments of a chimeric protein facilitates their separation at low pH. The major requirement of this system is that the desired segment of interest is not acid-labile. Chimeric proteins comprising hormones such as insulin and somatostatin have been cleaved with cyanogen bromide, which is specific for the carboxyl side of methionine residues, to release the desired hormone. This method is not suitable when the desired protein contains methionine residues.
Cleavage of chimeric proteins by site-specific proteolysis has also been investigated. Chimeric proteins into which a chicken pro alpha-2 collagen linker was inserted could be specifically degraded by purified microbial collagenase to release the components of the chimeric protein. Use of proteolytic enzymes to cleave the chimeric protein has drawbacks because the enzymes can be expensive, the yield of product is frequently low, and it can prove difficult to later separate the enzyme (a protein) from a desired cleavage product. Other methods for purification and recovery of a desired recombinant protein include construction of a poly-arginine tail at the carboxyterminus of the protein. The arginine residues increase the overall basicity of the protein which facilitates purification of the desired protein by ion exchange chromatography. Subsequent removal of the poly-arginine tail by carboxypeptidase B regenerates the desired protein and allows purification from basic contaminants due to the reduction in pI of the desired protein.
Acid cleavage can be accomplished by placing a specific dipeptide at the junction of the leader sequence and the peptide. Selection of the second amino acid will determine the rate at which the dipeptide bond is cleaved under acidic conditions. Of course, if the desired peptide contains any internal dipeptide sequences that are acid cleavable, then the cleavage site at the junction of the leader and the peptide must undergo acid cleavage at a substantially greater rate than the internal cleavage in order to avoid unacceptable loss of yield.
In addition to difficulties encountered with chimeric protein cleavage, natural amino acid modifications such as C-terminal amide group substitution, performed routinely in vivo, are difficult to perform in vitro. These post-translational modifications often result in the most potent or longest acting form of a peptide and render the peptide most suitable for pharmaceutical use. For many peptides, C-terminal amidation is important for biological activity. However, recombinant expression systems for large scale production of active peptides cannot easily carry out the necessary C-terminal modification.
Carboxypeptidase enzymes are known to catalyze transpeptidation reactions, yielding C-terminally amidated peptides. However, wild type carboxypeptidase enzymes are not useful for C-terminal amidation of many peptides. For example, the inherent substrate specificity of wild-type carboxypeptidase restricts the variety of peptides that may be modified using this enzyme. Transpeptidation occurs when an amino acid or amino acid derivative acts as a leaving group and the nucleophile is an amino acid, or amino acid derivative, such as an amino acid ester or amino acid amide. “Transamidation” includes transpeptidation, in that an amide bond is formed between the nucleophile and the peptide substrate. However, in a transamidation reaction, the nucleophile is not necessarily an amino acid.
In particular, carboxypeptidase Y displays a strong preference for peptides with a penultimate apolar residue. Substrates having a penultimate amino acid with a positively charged side chain are not effectively hydrolyzed nor transacylated by carboxypeptidase Y. For example, the substrate FA-Arg-Ala-OH (SEQ ID NO:1) is hydrolyzed about 500 times more slowly than the substrate FA-Leu-Ala-OH (SEQ ID NO:2). Unfortunately, the amino acid sequences of many pharmaceutically important peptides, including growth hormone releasing factor (GRF) or glucagon like peptide-1 (GLP-1), have a penultimate or ultimate amino acid with a positively charged side chain, making transamidation with carboxypeptidase Y commercially impractical.
U.S. Pat. No. 6,251,635 describes the treatment of a chimeric protein, including multiple copies of a target sequence, in a precursor peptide which includes hCA-(MetValAspAspAspAspAsn-ECF2)n-Xxx (SEQ ID NO:3), where hCA is human carbonic anhydrase, ECF2 is a polypeptide fragment having the formula: Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro (SEQ ID NO:4); and Xxx is typically a C-terminal carboxylic acid (“—OH”), a C-terminal carboxamide (“—NH2”), or group capable of being converted into a C-terminal carboxamide, such as an amino acid residue or a polypeptide group (typically having from 2 to about 10 amino acid residues), and n is an integer (typically 2 to 20). Such a precursor peptide may be treated with CNBr to form ValAspAspAspAspAsn-ECF2-Hse (SEQ ID NO:5) peptide fragments (where Hse is a homoserine residue produced by the reaction of CNBr with a Met residue). The peptide fragments may then be reacted with a nucleophile such as o-nitrophenylglycine amide (“ONPGA”) in the presence of a peptidase such as carboxypeptidase Y resulting in the replacement of the Hse residue by ONPGA. Upon photolysis, the transpeptidation product is converted to a C-terminal carboxamide. The N-terminal tail sequence, ValAspAspAspAspAsn (SEQ ID NO:6), may be cleaved off the fragments by treatment with hydroxylamine.
Another method of forming a C-terminal amide on a recombinantly produced polypeptide uses the enzyme peptidyl alpha-amidating enzyme which is present in eukaryotic systems. The enzyme has been used to form an amide on the C-terminal amino acid of recombinantly produced peptides, like human growth hormone releasing hormone in vitro, as described by Engels, Protein Engineering, 1:195-199 (1987). While effective, the enzymatic method is time consuming, expensive, gives unpredictable yields, and requires significant post-reaction purification.
Patchomik and Sokolovsky, JACS, 86: 1206-1212 (1964) describe the reaction of peptidyldehydroalanine in acidic solution to yield an amidated peptide. It is however undesirable to employ this technique to amidate a peptide as it requires relatively harsh conditions, viz. boiling in mild acidic aqueous solution. Even in the presence of a Lewis acid catalyst such as Hg2+, the reaction still not very successful. (Edge and Weber, Int. J. Peptide Protein Res., 18: 1-5 (1981)). If the reaction substrate is treated with organic and/or inorganic acid and contains acid sensitive amino acid residues, the harsh reaction conditions will produce side products.
Thus, known polypeptide amidation processes suffer from numerous drawbacks. Such reactions may be sequence-specific, require harsh conditions and may require multiple steps for cleavage and amidation.
Accordingly, there is a need for an improved process that provides for the efficient cleavage and amidation of a peptide.